A prophylactic HIV-1 vaccine is urgently needed, and an antigen that elicits strong broadly reactive neutralizing antibody (nAb) responses against the viral envelope glycoprotein would be an important component. However, the challenges in vaccine development include the extraordinary genetic diversity and immune evasion capacity of HIV-1 and the limited availability of epitopes for broadly nAbs. Immunogens that mimic an important process in natural infection and initiate appropriate innate immune responses are expected to be advantageous. Virus-like particles (VLPs) represent an attractive approach to present Env in a form similar to its exposure on virions. We recently reported that modified Env proteins can be designed to be incorporated into VLPs at very high levels (Wang, 2007). We hypothesize that VLPs containing such modified Env will elicit enhanced nAb responses, and are proposing additional new approaches to generate VLPs with greater immunogenicity. One approach will focus on enhancing the incorporation of stable Env trimers into VLPs. Alternatively, VLP immunogenicity will be enhanced by incorporation of molecular adjuvants, including a membrane -anchored form of flagellin, the natural ligand of the toll-like receptor 5 (TLR5), or a membrane- anchored form of GM-CSF. We expect that the resulting VLPs will elicit improved neutralizing antibody responses, as well as cell-mediated cellular immunity. We hypothesize that these novel VLPs will elicit higher titers of broadly reactive nAbs and enhanced mucosal immune responses when administered by mucosal inoculation, and we will test this hypothesis in a guinea pig model. Because DNA vaccines are effective in priming antibody responses as well as eliciting cellular responses (Singh et al., 2005), as a comparison we will also generate DNA constructs expressing clade B and C Env and Gag for use in priming by DNA vaccines, including a cross-clade DNA prime-VLP boost regimen to focus cross-clade immunity. Our goal is to develop an HIV vaccine that is effective at reducing the incidence of infection at mucosal surfaces. The combination of Env antigen presentation in its native state combined with a membrane-anchored adjuvant and mucosal delivery provides a promising approach to stimulate strong nAb response against HIV, designed to block the initial step in the infection process. Because of the relatively low efficiency of HIV transmission at mucosal surfaces, even a modest enhancement of protective mucosal responses could have a significant effect on reducing disease incidence. Among the possible outcomes which may result in such enhanced protection are: increasing the magnitude and/or duration of the response, increasing the breadth of cross-reactivity of the responses, and enhancing the levels of responses at local mucosal sites. Despite tremendous efforts, an effective vaccine for AIDS prevention remains elusive and novel approaches are needed. Our research is focused on developing novel particulate antigens that will elicit broadly reactive neutralizing antibody responses as well as cell-mediated immunity. We will also focus on enhancing immune responses at mucosal surfaces, where most HIV infections are transmitted.